Project Summary/Abstract Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated colitis and is responsible for significant morbidity, mortality and increased healthcare costs. Antibiotics disrupt the indigenous gut microbiota, reducing resistance to C. difficile colonization. Our knowledge of the mechanism(s) by which the gut microbiota confers resistance to CDI is incomplete, presenting a significant roadblock to improving preventative and therapeutic approaches against this pathogen. My long-term goal is to understand how the gastrointestinal tract microbiota mediates colonization resistance against enteric pathogens, including C. difficile. The overall objective of this application is to define members of the gut microbiota that are able to alter bile acids and consume sugars which are required for C. difficile colonization and pathogenesis. Based on preliminary studies the central hypothesis is that the production and consumption of specific metabolites (secondary bile acids and sugars) by the indigenous gut microbiota contribute to colonization resistance against C. difficile. The rationale that underlies the proposed research is that the targeting of metabolites required for C. difficile colonization has the potential to improve directed therapeutic approaches for this infection. Guided by strong preliminary data, this hypothesis will be tested by exploring the following key questions: 1) Can restoring microbial-mediated secondary bile acid metabolism in the large intestine restore colonization resistance against C. difficile? and 2) Can restoring bacteria that are able to compete for the same nutrients (sugars) as C. difficile requires for growth reestablish colonization resistance against C. difficile? To answer the first key question, we will select for and characterize bacteria that are capable of secondary bile acid metabolism. Genetic engineering of bacterial strains for efficient enzyme delivery to the gastrointestinal tract will be evaluated in vitro and in vivo, in a mouse model of CDI. Under the second key question, we will screen and characterize bacteria that are able to compete for the same nutrients as C. difficile. Bacteria will be evaluated by competition assays in vitro and in vivo, in a mouse model of CDI. Both approaches will explore how these bacterial strains alter C. difficile colonization resistance in the gastrointestinal tract as well as how they alter the surrounding environment including the microbiome, metabolome and host response. The proposed research program in this application is innovative because it represents a departure from the status quo, namely in the approach of using a targeted bacterial therapy to restore secondary bile acids and competition, ultimately restoring colonization resistance against C. difficile. The proposed research is significant, because it will lead to the identification of bacteria and new-targeted approaches to be used for therapeutic interventions to prevent or treat CDI, and potentially other metabolic diseases.